Phosphorylation of nucleosides by P-N bond species generated from prebiotic reduced phosphorus sources

Abstract

P-N species e.g., amidophosphates readily phosphorylate organics, thereby overcoming the so-called 'Phosphate Problem'. However, the formation of amidophosphates by plausible early Earth geochemical pathways is limited. We herein show that ammonolysis of the prebiotically plausible dimer of phosphite, pyrophosphite, readily affords amidophosphite, the monomeric P-N derivative of phosphite. Amidophosphite then undergoes spontaneous oxidation to form monoamidophosphate (MAP) and diamidophosphate (DAP) at room temperature (yields of the inorganic P-N species up to 48%). Oxidation of amidophosphite is promoted by O₂, H₂O₂, ClO⁻ and by UV light irradiation (365 nm). Both amidophosphite and MAP and the crude reaction mixture react with nucleosides to form nucleotides with both phosphate and H-phosphonate (yields up to 65%) at 80 °C in the presence of urea, showing that monoamidated phosphorus compounds also willingly promote prebiotic reactions. This observation expands the range of P-N phosphorylating agents that can play a role in the chemical evolution of prebiotic molecules on the early Earth.

Fig. 1. Time progression ³¹P-NMR {H-coupled} spectra showing the formation of P-N compounds over time.

The reaction was carried out by adding 4 ml of 14 N ammonium hydroxide (pH = 14) to a clean glass vial (20 ml capacity) containing powdered (0.2 g) pyrophosphite and a clean magnetic stirring bar. The (Teflon capped) sealed reaction vial was immediately stirred at room temperature under air. Inset is ×1.5 magnification (left) and ×2 magnification (right).

Scheme 1. Routes to form various P-N, P-O, P-O-P and P-H species obtained*.

*Reaction pathway to form P-N compounds by pyrophosphite ammonolysis with 14 N NH₄OH to form various P-N species and various other inorganic P-compounds.

Fig. 2. Investigation of N incorporation in pyrophosphite.

a The fraction of P-H bonds in solution vs. mole fraction of N in solution stays roughly constant, showing little oxidation of phosphite, b the fraction of P-N bonds in solution vs. the mole fraction of N in solution increases with increasing N in solution, and c the fraction of P-H stays roughly constant even though the number of P-N bonds increases.

Fig. 3. ³¹P-NMR {H-coupled} spectra showing the formation of MAP and DAP by the oxidation of amidophosphite produced by the ammonolysis pyrophosphite by hypochlorite and peroxide under anoxic conditions.

In each reaction, 4000 µl, 14 N NH₄OH and 1.05 mmoles of pyrophosphite were used. The initial molarities and volumes of the respective oxidants are (a) No additive, (b) 0.70 M ClO⁻ (300 µl), (c) 0.70 M ClO⁻ (700 µl), (d) 4.9 M H₂O₂ (300 µl) and (e) 4.9 M H₂O₂ (700 µl). For the complete figure see SI Fig. S18). Inset is ×1.5 magnification (left) and ×3 magnification (right).

Fig. 4. Various sources of reduced P were used as starting material to form pyrophosphite.

Pyrophosphite was obtained generally by heating the aqueous solutions of equimolar (0.1 M) phosphorus acid and sodium hydroxide at 200 °C, in a tube furnace, in an atmosphere of N₂. Hypophosphite was reacted with two equivalents of H₂O₂ and one of FeCl₂ in water to form phosphite alongside other oxidation (inorganic) P products. The aqueous solutions of all sources of phosphite were heated at 78–80 °C, un-sealed, in the presence of urea to form pyrophosphite.

Fig. 5. ¹H-NMR spectra of crude reaction of cytidine and the phosphorylating agents after 3 days.

(a) t = 15 min of reaction, (b) MAP, (c) DAP, (d) Phosphate and (e) Pyrophosphate. (For conditions see Table 2 and for full spectra see Fig. S46).

Fig. 6. Reaction mixture of deoxycytidine (dC) (0.05 mmol, 1 equiv), pyrophosphite (5 equiv), urea (3 equiv) and 100 μL of water at 80 °C after 3rd day.

The reactions were carried out at 80 °C for 3 days uncapped. a ¹H NMR of reaction crude, red color means dC (starting material), the signals of products are close to the dC signal. b ³¹P NMR {H-Coupled} of the same reaction, after the 3rd day, pyrophosphite was hydrolyzed to phosphite and oxidized to phosphate, the products are 5’-H-phosphonate deoxycytidine, 3’-H-phosphonate deoxycytidine and the 3’,5’-H-diphosphonate deoxycytidine.

Fig. 7. Mass spectra of the crude reaction of uridine (0.05 mmol, 1 equiv), amidophosphite (crude reaction mixture) (5 equiv), urea (3 equiv) and 100 μL of water after 3 days at 80 °C.

The reactions were carried out at 80 °C for 3 days uncapped. The exact mass of the 5’-H-phosphonate is 307.0337 m/z [M-H]⁻, found 307.0401 m/z [M-H]⁻, and the exact mass of the 2’ or 3’,5’-H-bisphosphonate is 371.0051 m/z [M-H]⁻, found 371.0113 m/z [M-H]−.